Purdue University
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Birck Nanotechnology Center
2-2009
Capture and alignment of phi29 viral particles in
sub-40 nanometer porous alumina membranes
Jeong-Mi Moon
Purdue University - Main Campus
Demir Akin
Birck Nanotechnology Center, Purdue University
Yi Xuan
Birck Nanotechnology Center, Purdue University
P. D. Ye
Birck Nanotechnology Center and School of Electrical and Computer Engineering, Purdue University, yep@purdue.edu
Peixuan Guo
University of Cincinnati - Main Campus
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Moon, Jeong-Mi; Akin, Demir; Xuan, Yi; Ye, P. D.; Guo, Peixuan; and Bashir, Rashid, "Capture and alignment of phi29 viral particles in
sub-40 nanometer porous alumina membranes" (2009). Birck and NCN Publications. Paper 366.
http://docs.lib.purdue.edu/nanopub/366
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Authors
Jeong-Mi Moon, Demir Akin, Yi Xuan, P. D. Ye, Peixuan Guo, and Rashid Bashir
This article is available at Purdue e-Pubs: http://docs.lib.purdue.edu/nanopub/366
Biomed Microdevices (2009) 11:135–142
DOI 10.1007/s10544-008-9217-0
Capture and alignment of phi29 viral particles in sub-40
nanometer porous alumina membranes
Jeong-Mi Moon & Demir Akin & Yi Xuan & Peide D. Ye &
Peixuan Guo & Rashid Bashir
Published online: 4 September 2008
# Springer Science + Business Media, LLC 2008
Abstract Bacteriophage phi29 virus nanoparticles and its
associated DNA packaging nanomotor can provide for
novel possibilities towards the development of hybrid bionano structures. Towards the goal of interfacing the phi29
viruses and nanomotors with artificial micro and nano-
J.-M. Moon : Y. Xuan : P. D. Ye
Birck Nanotechnology Center, School of Electrical and Computer
Engineering, Purdue University,
West Lafayette, IN 47907, USA
D. Akin
Birck Nanotechnology Center,
Weldon School of Biomedical Engineering, Purdue University,
West Lafayette, IN 47907, USA
P. Guo
School of Biomedical Engineering, University of Cincinnati,
Cincinnati, OH 45221, USA
R. Bashir (*)
Micro and Nanotechnology Laboratory, Department of Electrical
and Computer Engineering and Bioengineering,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801, USA
e-mail: rbashir@uiuc.edu
Present address:
J.-M. Moon
Micro and Nanotechnology Laboratory,
Department of Electrical and Computer Engineering,
University of Illinois at Urbana-Champaign,
Urbana, IL 61801, USA
Present address:
D. Akin
Center for Cancer Nanotechnology Excellence,
Stanford University School of Medicine,
The James H. Clark Center,
Stanford, CA 94305-5427, USA
structures, we fabricated nanoporous Anodic Aluminum
Oxide (AAO) membranes with pore size of 70 nm and
shrunk the pores to sub 40 nm diameter using atomic layer
deposition (ALD) of Aluminum Oxide. We were able to
capture and align particles in the anodized nanopores using
two methods. Firstly, a functionalization and polishing
process to chemically attach the particles in the inner
surface of the pores was developed. Secondly, centrifugation of the particles was utilized to align them in the pores
of the nanoporous membranes. In addition, when a mixture
of empty capsids and packaged particles was centrifuged at
specific speeds, it was found that the empty capsids deform
and pass through 40 nm diameter pores whereas the
particles packaged with DNA were mainly retained at the
top surface of the nanoporous membranes. Fluorescence
microscopy was used to verify the selective filtration of
empty capsids through the nanoporous membranes.
Keywords phi29 . Nanoporous membrane .
Atomic layer deposition . Alignment . Capture
1 Introduction
Viruses are masterpieces of nanoengineering designed as
replicating machines. The novelty and ingenious design of
these biological nanomachines can help inspire the development of biomimetics for nanodevices. For instance,
the bacteriophage phi29 contains a switchable DNApackaging motor that has been shown to be one of the
most powerful biomotors studied so far (Shu and Guo
2003a). This nanomotor is driven by six ATP-binding
pRNA molecules that bind to the connector protein, as
schematically shown in Fig. 1 (Guo et al. 1991; Shu and
Guo 2003b). Conformational changes and sequential action
136
Biomed Microdevices (2009) 11:135–142
2 Experimental
2.1 Material and reagents
High-purity Al foil (99.999%, 0.2 in. thickness) was purchased
from Research and PVD Materials. Trimethylaluminum
(TMA) and 3-(trimethoxysilyl)propyl aldehyde (TMSPA) were
purchased from Aldrich and used without further purification.
Deionized water was obtained from an ultrafiltration system
(Milli-Q, Millipore) with a measured resistivity above
18 MΩcm and passed through a 0.22 μm filter to remove
particulate matter.
Fig. 1 Schematic of a phi29 viral particle. The particle size is about
40×50 nm and contains a DNA packaging nanomotor at one end
of the pRNA ensure the motor rotates continuously, using
ATP as the energy source to package DNA molecules that
are up to 20,000 base pairs or 6 μm in length (Zhang et al.
1998; Chen and Guo 1997; Guo et al. 1998; Chen et al.
1999; Guo et al. 1987). This packaging process involves a
delicate balance of forces which are both mechanical and
electrostatic in nature (Klug et al. 2005). The motor of
phi29 can generate a force of up to 57 pN (Smith et al.
2001). Such a force is enough to pump DNA against the
high internal pressure that builds up as the viral DNA is
packed inside the capsid.
For the separation, collection, and filtration of viruses,
membrane-based technologies have been developed as
useful and efficient methods (Lakshmi and Martin 1997;
van Reis and Zydney 2001; Xu et al. 2005; Yang et al.
2006; Lee et al. 2002). Potentially, membranes are well
adapted for these applications because their pore size and
surface chemistry can be adjusted to the size and surface
chemistry of an organism of interest, and the specific
capture (selective sensing) of this organism could be
possible. Several types of membranes have been employed
for virus capture and separation; however, many of these
have not been effective (van Voorthuizen et al. 2001; Otaki
et al. 1998; Urase et al. 1996). For example, in case of
microfiltration membranes, the pore size is typically much
larger than the size of the virus particles that are tens of
nanometers in size (van Voorthuizen et al. 2001; Otaki et al.
1998). The smaller pore size ultramembranes have uneven
distribution of pore size, allowing virus particles to
permeate into a small number of abnormally large pores
(Urase et al. 1996). Although chemically specific filtration
using nanoporous membranes has been shown for small
molecules (Jirage et al. 1999; Lee and Martin 2001;
Fernandez-Lopez et al. 2001; Chun and Stroeve 2002),
specific bio-organism immobilization, capture, and detection remain to be a great technical challenge.
2.2 Fabrication of nanoporous AAO membrane
Aluminum foil was first anodized in a 0.3 M oxalic acid
solution at 5°C at a constant applied voltage of 40 V for
20 h and then etched in an aqueous mixture of 6 wt %
phosphoric acid and 1.8 wt % chromic acid at 60°C. A
second anodization was performed for 8 h using the same
conditions as above, followed by dissolution of the
remaining Al in a saturated HgCl2 solution. Pore widening
and removal of the barrier oxide layer were carried out by
chemical etching in 0.8 wt % phosphoric acid at 30°C for
60 min to produce AAO membranes with highly ordered
pores (diameter = 75 nm, center-to-center distance =
105 nm), as shown in Fig. 2(a) and (b). The final thickness
of the membrane was 24 μm with pores across the
membranes.
2.3 Atomic layer deposition
We used an ASM F-120 ALD system for shrinking the pore
size of AAO membrane. ALD Al2O3 films were deposited
by using alternating pulse of trimethylaluminum (TMA) [Al
(CH3)3] and water (H2O) precursors. H2O vapor was used
to oxidize chemisorbed TMA. N2 was used as a carrier gas
to transport precursor vapor to the membrane surface and
purge reaction gases from the reactor during each halfreaction. The pulse time of TMA, H2O and N2 were 0.8 s,
1.2 s and 2.0 s, respectively. For our purpose, the deposition
process was run for 133 to 211 cycles at 2–5 Torr at 300°C
and the growth rate was 0.9 Å per cycle for Al2O3.
2.4 Array of phi29 procapsid on the alumina membranes
3% 3-(trimethoxysilyl)propyl aldehyde (TMSPA) solution
was prepared in ethanol/H2O (95%/5%) and membranes
were immersed to clean the ALD/AAO membrane for 3 h.
After washing the aldehyde-silanized ALD/AAO with
ethanol several times, the membranes were dried in a
stream of nitrogen gas and heated at 110°C for 20 min. The
aldehyde-silanized alumina membrane was incubated in a
Biomed Microdevices (2009) 11:135–142
Fig. 2 Field-emission scanning
electron microscope (FE-SEM)
images of the anodic aluminum
oxide (AAO) membranes and
further shrunk by atomic-layer
deposition (ALD). The images
show the shrunk pore sizes; (a)
and (b) 75±5 nm as anodized
nanoporous membranes, (c) and
(d) 39±4 nm, (e) and (f) 25±
3 nm, and (g) and (h) 15±1 nm
137
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
solution of procapsid (without pRNA; 0.01 mg/mL in TMS
buffer) for 15 h at room temperature. The membrane was
then immersed into fresh TMS buffer for 30 min. at room
temperature to remove any unattached procapsids.
(10 mM in TMS), 6 μL of gp16 (0.5 mg/mL), and 1 μL of
DNA-gp3 (200 ng/μL) and incubated 60 min at room
temperature to complete the DNA-packaging reaction. We
used these DNA-packaged phi29 particles for the experiments described in subsequent sections.
2.5 Procedure for the DNA-packaging of phi29 in vitro
Briefly, 1 μg of RNA in 1 μL RNase free H2O was mixed
with 10 μL of purified procapsids (0.4 mg/ml) in TMS
(100 mM NaCl, 10 mM MgCl2, 50 mM Tris, pH 7.8) and
then incubated for 60 min at room temperature. The pRNAenriched procapsids were then mixed with 3 μL of ATP
3 Results and discussion
In this paper, we report the use of anodized aluminum
oxide (AAO) membranes with narrow pore size distribution
and chemically modifiable surfaces for alignment and
138
Biomed Microdevices (2009) 11:135–142
capture of phi29 particles. Highly ordered nanoporous
AAO membranes were prepared by a two-step anodization
process similar to described above and also previously
(Moon and Wei 2005), resulting in structures as shown in
Fig. 2(a) and (b).
The nanopores of these AAO films are still big
compared to the phi29 nanomotors. Next, we decreased
the pore sizes using atomic layer deposition (ALD) which
significantly shrinks but does not fully seal the nanopores
(Xiong et al. 2005; Miikkulainen et al. 2008). ALD is a
promising technique to form ultrathin films uniformly over
a large wafer and 3-D structures such as deep trenches and
deep holes with atomic-scale thickness controllability. ALD
consists of two half reactions: (1) self-limiting chemisorption of metal-organic or metal-halide precursor on the wafer
surface and (2) reaction of the chemisorbed these species
with oxygen-containing species such as water vapor, O2,
O3, or metal alkoxide. Generally ALD-growth is carried out
at a relative low temperature (ca. 300°C) because thermal
decomposition of precursor at higher temperatures deteriorates the thickness controllability. After the ALD process
was completed, field-emission scanning electron microscope (FE-SEM; Hitachi S-4800) analysis of nanoporous
ALD/AAO membranes revealed pore diameters on the
order of 39 nm, 25 nm, and 15 nm, respectively, with
increasing times used for the deposition (Fig. 2(c)–(h)).
To use the ALD/AAO membranes for the biochemically
assisted self-assembly, biomolecules need to be attached to
Fig. 3 (a) Schematic depiction
of the experimental procedure, (b)
transmission electron microscopy
(TEM) of the empty phi29 procapsids, (c) expanded view of the
region in dashed circle in (b) to
show the empty procapsid particles (black arrow) and the connectors (white arrow)
the surface of the alumina membrane. The experimental
procedure of the immobilization on the membrane surface
and hybridization of bio-nano structures is schematically
shown in Fig. 3(a). The procapsid of phi29 is formed by
self assembly via protein–protein interactions of the
scaffolding protein gp7, the capsid protein gp8, and
the portal vertex protein gp10 (Lee and Guo 1995). The
procapsids contain a connector (or portal vertex) with 12
protein subunits, a circular oligomer with a central tunnel
through which the DNA is translocated into the procapsid
during the assembly process or ejected out of the mature
capsid during the host cell infection. Figure 3(b) and (c)
shows tunneling electron microscopy (TEM, Philips C-100)
images of negative-stained procapsids with an average
diameter of 45 nm as well as unreacted connectors with an
average diameter of 14 nm. These connectors could be
removed with further purification of the samples.
For the array of procapsid on the alumina membranes,
3-(trimethoxysilyl)propyl aldehyde (TMSPA), was used as
a bi-functional modifier to functionalize the AAO membrane. This is possible since the silane groups can form
covalent bonds with the Aluminum Oxide (Al2O3) surfaces
and the amine groups of procapsid can be attached to the
aldehyde groups on the membranes. First, the membrane
surface (39 nm pore diameter) was modified with TMSPA.
The aldehyde-silanized alumina membrane was then incubated in a solution of procapsid (without pRNA). The
morphology of biomaterial particles on the ALD/AAO was
(a)
39~ 15 nm
silanize
Procapsid
ALD/AAO nanotemplate
Polish
0.06- 0.02 µm
Red-Final C
Procapsid
(b)
200 nm
(c)
Biomed Microdevices (2009) 11:135–142
139
characterized by field-emission scanning electron microscope (FE-SEM; Hitachi S-4800). Metal Au/Pd film was
sputter deposited on the surface of biomaterials for FESEM samples. Binding of procapsid particles both in the
pores and on the top surface of the membrane with MPTMS
are shown in the FESEM images in Fig. 4(a) and (b). The
particles have an average size of 30 nm as shown in
Fig. 4(c). The large size distribution is attributed to a
mixture of procapsid and individual connector particles in
the solution.
In order to attach the biomaterial particles to the pores
only and not to the top surface, the aldehyde-silanized
ALD/AAO membranes were polished for 10 s with
0.06 μm polishing cloth (Red-Final C; Allied High Tech
Products) to remove the aldehyde-silanized layer from the
face of the alumina surface. After polishing, the membrane
was placed into a solution containing 0.01 mg/ml of
procapsid dispersed in the TMS buffer for 4 h. The
resulting FESEM images in Fig. 4(d, e) show that indeed
the particles were mostly attached in the pores. The
(a)
(d)
(b)
(e)
25
200
(c)
Number of Procapsid
30
Count of procapsid
Fig. 4 (a and b) Field-emission
scanning electron microscope
(FE-SEM) images of the empty
phi29 particle binding to an
unpolished ALD/AAO membrane. (c) graph showing the
average size of the empty particle being 30 nm. The large size
distribution could be attributed
to a mixture of procapsid, protein fragments, and individual
connector particles in the solution. (d and e) FE-SEM images
of the empty phi29 particle
binding to a polished membranes. (f) Histogram showing
that particle binding to the unpolished membrane occurs 50%
in the pores and 50% out of the
pores. In contrast, the particle
binding to the polished membrane results in 84% in the pores
and 16% out of the pores
histogram in Fig. 4(f) represents the quantity of particles
attached to the membranes. The unpolished membrane
shows that 50% of the biomaterial particle binding occurred
in the pores and 50% out of the pores. In contrast, the
polished membrane shows particle binding to be localized
at about 84% in the pores and about 16% out of the pores.
For the size selective capture of biomaterial onto ALD/
AAO membranes, we also filtered the solution with the
phi29 particles by centrifugation. The membrane pore size
can be adjusted to smaller than that of a biomaterial particle
allowing the specific capture of this particle of interest. To
investigate this hypothesis, we decreased the pores to
38 nm on as-prepared AAO substrates using the ALD
method. First, we tested the ability of the membranes to
capture empty procapsids. Then, we attempted to use DNApackaged phi29 particles with the nanoporous ALD/AAO
membranes. The procedure for the DNA-packaging of
phi29 in vitro was used as described in the earlier section
and previously (Lee and Guo 1995). Figure 5(a, b) shows
the TEM images, negatively stained, of the DNA filled
20
15
10
5
0
10
20
30
40
Procapsid length / nm
50
(f)
in pores
out of pores
150
100
50
0
without
polishing
with
polishing
140
Fig. 5 (a) Transmission electron microscopy (TEM) of the
DNA filled phi29 capsids, (b)
Expanded view of the region in
dashed circle in (a) to show the
DNA packaged particles. Note
the difference in contrast between the empty and filled particles. The packaged capsids
have greater electron density
resulting in clearer contrast.
Field-emission scanning electron microscope (FE-SEM)
images of the top (c) and bottom
(d) of the ALD/AAO membrane
after centrifugation with empty
procapsids. As can be noted,
very few particles remain on the
top surface and most of the
particles have passed through
and are retained on the bottom
side. Field-emission scanning
electron microscope (FE-SEM)
images of the top (e) and bottom
(f) of the ALD/AAO membrane
after centrifugation with DNA
filled capsids. More particles are
retained on the top which do not
pass through
Biomed Microdevices (2009) 11:135–142
(a)
(b)
200 nm
(c)
(d)
(e)
(f)
phi29 capsids. In clear contrast to the TEM images of the
empty procapsids, the regions inside the particles appear
dense and brighter for the packaged capsids. We then
performed filtration experiments in a modified swinging
bucket centrifuge (Eppendorf 5804) at 1680 x g (3000 rpm)
for 5 min. The empty procapsid particles penetrated through
the ALD/AAO membrane pores and were retained on the
bottom surface of the membrane, as shown in Fig. 5(c) (top
surface) and (d) (bottom surface). This novel result is
particularly interesting considering that the membrane pores
(∼38 nm) are smaller than the procapsid particles (average
diameter of ∼ 45 nm). However, lots of particles were
observed on the top side of membranes for the case of the
packaged capsid particles filtered through the ALD/AAO
membrane (Fig. 5(e), (f)).
To identify and also characterize their DNA encapsulation status, the particles found on both sides of the
membrane in the Fig. 5(e) and (f), top and bottom,
respectively, were dual stained with DiI (stains proteins
fluorescent red) and YoYo-1 (stains DNA fluorescent
green-blue) according to an already published protocol
(Akin et al. 2004), followed by fluorescence image
acquisition of both surfaces using a color, cooled CCD
camera (Fig. 6(a), (b)). As seen in the Fig. 6(a) (top side)
and (b) (bottom side), the top surface showed yellow colocalization signal of fluorescent green to light blue labeled
DNA and fluorescent red phi29 procapsid. In contrast, the
bottom surface showed mainly the red-labeled empty
procapsid particles and green to light blue DNA as separate
components. Further analysis of the RGB channels of
Fig. 6(a) and (b) was done by plotting the intensity profiles
of each color channel of the top (Fig. 6(c)) and the bottom
(Fig. 6(d)) of the membranes. The resulting separate color
channel intensities of the top and the bottom surfaces were
quantified (Fig. 6(e)) and it was found that the majority of
the DNA (green) was retained within the capsids (red)
located at the top surface. Some DNA fluorescence was
also observed in the images acquired from the bottom side
indicating the possible existence of partially filled capsids
or free DNA since these could also pass through the pores.
These results indicate that the nanoporous ALD/AAO
membranes with appropriate centrifugation conditions
Biomed Microdevices (2009) 11:135–142
141
(a)
(c)
(b)
Top
(e)
100
Bottom
90
Top
(d)
Bottom
Mean Intensity
80
70
60
50
40
30
20
10
0
Capsid (R)
DNA (G)
Fig. 6 Fluorescence images of phi29 capsids on top side (a) and the
bottom side (b) of the membrane after the centrifugation process. High
resolution view of the fluorescence image is shown in the inset of (a)
and (b), respectively. The top surface shows yellow co-localization
signal of fluorescent green to light blue labeled DNA and fluorescent
red capsid. In contrast, the bottom surface shows mainly the redlabeled empty procapsid particles. Average intensity values for each of
the RGB channels of the top surface (c) and the bottom surface are
shown in (d) as extracted from the line profile plots (blue lines in a and
b). Quantification of the locations of red fluorescent capsid and green
fluorescent DNA are shown in (e). Majority of the DNA-containing
capsids are found on the top surface, however, some partially packaged
capsids are also evident at the bottom surface as indicated by the
observation of green fluorescence signal at the bottom surface
could be used for discrimination of phi29 particles with and
without fully packaged DNA from a mixture and this novel
method may be of use for the determination of DNA
packaging rate as an alternate method to plaque/infectivity
titration based methods.
We suggest that the empty procapsid undergoes structural
changes due to centrifugal force (1680 x g) during the
centrifugation based filtering. The capsids have thin walls
compared to their diameters, for example, the wall thickness of
the phi29 capsid is ∼1.5 nm, whereas its linear dimensions are
on the order of 40–50 nm (Tao et al. 1998). Moreover, the
protein: protein interactions within the empty procapsids may
not be fully stabilized. These factors might allow the empty
procapsid the flexibility to deform sufficiently to traverse
through a pore smaller than its own physical dimensions.
Many important and also related aspects of bacteriophage phi29 morphogenesis and mechanoelastic properties
have not been fully investigated yet. One of these aspects is
the influence of the enclosed viral DNA on the mechanical
properties of the viral particle and its stability. The prohead
of phi29 has 10 hexameric units in its cylindrical equatorial
region, and 11 pentameric and 20 hexameric units comprise
icosahedral end-caps with T=3 quasi-symmetry (Tao et al.
1998). In a recent study, under identical conditions, the
comparison of the spring constants of the empty capsid and
the viron of minute virus of mice (MVM), known to have
icosahedral (T=1) symmetry, showed that the presence of
the genomic DNA leads to an increase of the particle
stiffness by 3%, 42%, and 140% when probed along
fivefold, threefold, and twofold symmetry axes, respectively (Carrasco et al. 2006). The DNA molecule could
reinforce the particle by coating the internal surface of the
capsid, thus increasing the effective capsid wall thickness
homogeneously. By structural analogy, we believe this
phenomenon may also explain why DNA-packaged phi29
did not traverse the AAO membrane pores in our case;
however, more detailed studies are needed to fully
characterize this effect.
142
4 Conclusions
Our studies lay the ground work for interfacing viral
nanoparticles with the nanoporous AAO membranes. We
used atomic-layer deposition to precisely shrink the pore
size of the nanoporous alumina membranes down to 15 nm.
In addition, the alignment of viral particles on the pores was
demonstrated either by chemical functionalization and
polishing or by a centrifugation process. In particular, the
nanoporous ALD/AAO membranes can also be used to
separate fully DNA-packaged and unpackaged phi29
particles from a mixture by the centrifugation method.
The centrifugation while using the nanoporous membranes
can be used for the filtration, purification, and concentration
of various viruses.
Acknowledgments We acknowledge the funding from the National
Institutes of Health through the NIH Roadmap for Medical Research
(PN2 EY 018230 Nanomedicine Development Center), and NIH R21
EB007474 FE-SEM images were taken at the Birck Nanotechnology
Center Microscopy Facility. We also thank Dr. D. Sherman for the
TEM micrographs.
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